MII Eggs Research Articles

Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes†.

Numerous reference genes for use with quantitative reverse transcription polymerase chain reaction (RT-qPCR) have been used for oocytes, eggs, and preimplantation embryos. However, none are actually suitable because of their large variations in expression between developmental stages. To address this, we produced a standardized and merged RNA sequencing (RNAseq) data set by combining multiple publicly available RNAseq data sets that spanned mouse GV oocytes, MII eggs, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos to identify transcripts with essentially constant expression across all stages. Their expression was then measured using RT-qPCR, with which they did not exhibit constant expression but instead revealed a fixed quantitative relationship between measurements by the two techniques. From this, the relative amounts of total messenger RNA at each stage from the GV oocyte through blastocyst stages were calculated. The quantitative relationship between measurements by RNAseq and RT-qPCR was then used to find genes predicted to have constant expression across stages in RT-qPCR. Candidates were assessed by RT-qPCR to confirm constant expression, identifying Hmgb3 and Rb1cc1 or the geometric mean of those plus either Taf1d or Cd320 as suitable reference genes. This work not only identified transcripts with constant expression from mouse GV oocytes to blastocysts, but also determined a general quantitative relationship between expression measured by RNAseq and RT-qPCR across stages that revealed the relative levels of total mRNA at each stage. The standardized and merged RNA data set should also prove useful in determining transcript expression in mouse oocytes, eggs, and embryos.

Effects of electroacupuncture on pregnancy outcome in poor ovarian response patients of kidney essence deficiency and undergoing in vitro fertilization-embryo transfer

To observe the effects of electroacupuncture (EA) on ovarian reaction, egg and embryo quality, as well as pregnancy rate in poor ovarian response (POR) patients of kidney essence deficiency and undergoing in vitro fertilization-embryo transfer (IVF-ET). Ninety-six patients who met the inclusion criteria were randomly divided into an EA group and a control group, with 48 cases in each group. Before IVF-ET, the patients in the EA group received EA, once daily, 2 or 3 treatments a week for 12 weeks. Before and after the treatment, traditional Chinese medicine (TCM) syndrome score and clinical pregnancy rate were assessed in two groups. The concentrations of serum follicle-stimulating hormone (FSH), luteinsing hormone, estradiol, progesterone and anti-mullerian hormone were detected by chemiluminescence; the contents of serum insulin-like growth factor-1, serum inhibin B (INHB) and Kisspeptin in follicular fluid were determined by enzyme linked immunosorbent assay (ELISA); the antral follicle counting (AFC) was detected by color Doppler ultrasonography; and the egg and embryo conditions were observed under microscope. Fourteen days after embryo transfer, the positive rate of serum hemchoriconic gonadotropin (HCG) and clinical pregnancy rate were calculated. After the treatment, the TCM syndrome score and level of serum FSH were reduced (P<0.05); the INHB in serum and AFC were increased (P<0.05) when compared with those before the treatment in the EA group. After the treatment, in comparison with the control group, the TCM syndrome score and level of serum FSH were lower (P<0.05); and the contents of serum INHB, AFC, the numbers of MⅡ eggs and high-quality embryos, as well as serum HCG positive rate were all increased (P<0.05) in the EA group. EA can relieve the clinical symptoms of TCM in POR patients of kidney essence deficiency and undergoing IVF-ET, increase the ovarian reserve, reduce the serum FSH level, and improve the content of serum INHB, and the quality of eggs and embryos. This therapy tends to improve the clinical pregnancy rate and clinical pregnancy outcome.

Cyclin-dependent kinase 6 (CDK6) as a potent regulator of the ovarian primordial-to-primary follicle transition.

Introduction: Ovarian follicle development requires tight coordination between several factors to initiate folliculogenesis to generate a mature and fertile egg. Studies have shown that cell cycle factors might contribute to follicle development, hover specific knowledge on individual CDKs and follicle activation has not been investigated. Among cell cycle regulators, CDK6 is a key player through binding to cyclin D resulting DNA synthesis and genome duplication. Interestingly, the CDK6 gene is differentially expressed in oocytes and granulosa cells from human primordial and primary follicles, which suggest a potential role of CDK6 in the primordial-to-primary transition. In this study, we investigated the potential regulatory role of CDK6 in progression of primordial to primary follicle transition using BSJ-03-123 (BSJ), a CDK6-specific degrader. Methods: In mouse ovarian in vitro culture, BSJ reduced the activation of primordial follicles, and reduced follicle development. As a next step, we examined the egg maturation read-out and found that BSJ-treated follicles matured to competent MII eggs with resumption of first meiosis, comparable with the control group. Results: Noteworthy, it appears that inhibition of CDK6 did increase number of apotoptic cells, articular in the granulosa cells, but had no impact on ROS level of cultured ovaries compared to control group, indicating that the cells were not stressed. Oocyte quality thus appeared safe. Discussion: The results of this study indicate that CDK6 plays a role in the primordial-to-primary transition, suggesting that cell cycle regulation is an essential part of ovarian follicle development.

OP-21 - Age-related alterations in the surface morphology of zona pellucida prevent sperm binding to oocyte and affect fertilization.

Mammalian fertility with aging is thought to decrease due to an increase in aneuploidy, abnormal mitochondrial function, and changes in the expression of oocytes. Although previous studies have reported that naturally aged over 8-month-old female mice have lower fertility, the mechanism of egg quality deterioration is still unknown. In this study, we focus on the effects of aging on ovarian tissue and fertility using young mice (2 months old) and aged mice (8 months old) and clarify the mechanism of age-related deterioration of egg quality. When we examined the fertility by natural mating, the number of pups produced by aged mice was significantly decreased. Histological analysis of ovaries displayed a decrease in the number of remaining follicles, and ovulated oocytes ovulated number by superovulation treatment was also significantly reduced in aged mice. Examination of the fertilization ability in vitro revealed a significant decrease in the fertilization rate in aged mice, whereas there was no difference in gene expression in MII eggs by RNA-seq. These results suggest that the age-related decrease in fertilization ability of mouse oocytes is not due to gene expression in the oocytes themselves, but to inhibition of sperm binding to surface of the zona pellucida.

5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Efficacy of artificial oocyte activation in patients with embryo developmental problems: a sibling oocyte control study.

To explore whether artificial oocyte activation (AOA) can improve embryo developmental potentiality and pregnancy outcomes for patients with a history of embryo developmental problem. This was a retrospective study and candidate patients with embryo development problems were collected. A total of 1422 MII eggs from the enrolled 140 patients were randomized divided equally into 2 groups, half for the AOA group (AOA), and the rest of sibling mature eggs for the control group (non-AOA). The patients were further divided into two subgroups: (1) the rate of good-quality day 3 embryos was 0% (group 1, n = 66); (2) the rate of good-quality day 3 embryos ranged from 1 to 30% (group 2, n = 74). In the early embryonic growth, there were no significant differences in the outcomes of AOA and non-AOA groups in terms of normal fertilization rates, cleavage rates, day 3 good-quality embryo rates and available blastocyst rates (72.7% vs. 79.3%, 97.4% vs. 98.0%, 20.1% vs. 19.7%, 6.6% vs. 8.4% in group 1, respectively; 77.7% vs. 81.9%, 98.1% vs. 97.0%, 25.8% vs. 22.1%, 9.6% vs. 9.3% in group 2, respectively). In the late embryonic growth, no significant differences were found in biochemical and clinical pregnancy rates, implantation rates, miscarriage rates, and live-birth rates (50.0% vs. 45.2%, 45.2% vs. 40.5%, 37.3% vs. 31.3%, 10.5% vs. 11.8%, 40.5% vs. 35.7%, respectively) between two groups. In addition, neonatal outcomes were similar in both the groups as well. Our study demonstrated that the AOA using ionomycin 1h after ICSI did not bring benefits to the early or late development of embryos derived from patients with a history of embryo developmental problems.

AN INTERPRETABLE MACHINE LEARNING MODEL FOR INDIVIDUALIZED PROTOCOL SELECTION AND GONADOTROPIN DOSE SELECTION DURING OVARIAN STIMULATION

To develop an interpretable machine learning model for individualized gonadotropin dose selection during controlled ovarian stimulation. Historical, de-identified electronic medical record (EMR) data was collected from 4 IVF clinics in the United States. Records were filtered for autologous, non-canceled IVF retrievals, resulting in 7,977 cycles started between 2014 and 2020. A multiple linear regression model was developed with cross validation and recursive feature elimination to predict the number of eggs and mature (MII) eggs retrieved using baseline parameters available prior to start of treatment. The predictor variables were then used to create a patient similarity model based on K nearest neighbors (KNN), an interpretable machine learning technique. After identifying the best performing distance metrics, neighbor weights, and number of neighbors, the model was used to predict the number of eggs and MII eggs retrieved by calculating the weighted average from the set of K neighbors most similar to the patient of interest. The performance of the KNN model was compared to linear regression in terms of R-squared (R2) and mean absolute error (MAE). The KNN model was then used to (a) query the K most similar patients, and (b) identify the optimal gonadotropin dose in terms of highest number of MII eggs retrieved. We developed linear regression and KNN models using patient age, BMI, diagnosis, AMH, AFC, number of previous IVF cycles, and parity. KNN achieved highest performance using the Manhattan distance, 50-80 similar patients, and distance-based neighbor weighting. The KNN model outperformed linear regression for eggs retrieved (R2: 0.43 vs. 0.39, MAE: 4.84 vs. 4.98) and for MII eggs retrieved (R2: 0.39 vs. 0.35, MAE: 4.01 vs. 4.11). We then investigated the application of these models for gonadotropin dose selection. Linear models indicated that gonadotropin dose is negatively correlated with MII eggs, which may in part reflect that poor-prognosis patients are prescribed higher doses. In contrast, the KNN model showed that 22% of patients had a concave dose response curve, in which there was an optimal dose that maximized the number of MII eggs. We developed a patient similarity model using K nearest neighbors. The model showed better accuracy than linear regression for predicting eggs and MII eggs retrieved, and allowed the evaluation of which starting dose maximized the number of MII eggs retrieved, which is not possible with a linear model. Future work will optimize techniques for matching similar patients and extend the modeling for protocol selection.

Ovulation induction with high progesterone levels may be more suitable for elderly patients with low ovarian response

BackgroundThe objective of this study was to explore the outcomes of using the progestin-primed ovarian stimulation (PPOS) protocol in aged infertile women. The patients recruited in the study had displayed a poor ovarian response (POR) in the first IVF/ICSI-ET cycles with the ultra-short gonadotropin-releasing hormone agonist (GnRH-a) protocols. Materials and methodsA self-controlled retrospective study was conducted to investigate the clinical outcomes of 117 aged infertile women who met the inclusion criteria. The patients were grouped into two; group B included patients who had displayed a poor ovarian response (POR) in the first IVF/ICSI-ET cycle with the ultra-short GnRH-a protocol. Group A was made up of patients who underwent the PPOS protocol in the second cycle. The study was done between January 2015 to May 2018 in the reproductive and genetic centre of integrated traditional and western medicine, Affiliated hospital of Shandong University of traditional Chinese medicine. Reproduction-related clinical outcomes in the two groups were compared. ResultsThere were no statistically significant differences in the serum levels of LH, E2, and P on the trigger day between group A and group B (P＞0.05). The number of follicles with a diameter > 14 mm was significantly higher in the PPOS protocol patients than in the ultra-short GnRH-a protocol group (4.83 ± 2.82 vs. 3.25 ± 2.53, P < 0.01). The duration and total dosage of gonadotropin of the PPOS protocol group were less than in the previous ultra-short GnRH-a protocol, although the statistical differences were not significant (P > 0.05). The number of eggs obtained in the PPOS group was significantly higher than that of the previous one (4.29 ± 3.11 vs. 2.76 ± 2.33, P < 0.05). The numbers of MII eggs, cleavage, 2 P N, transplantable embryos, and high quality embryos were higher in the PPOS protocol group than that in the ultra-short protocol group. However, the differences between the two groups in all the above parameters were not statistically significant (P > 0.05). The rate of high-quality embryos was significantly higher in the PPOS protocol group than in the ultra-short protocol group (38.61(100/259) vs. 32.02(65/203), P < 0.05). Although not statistically significant (P > 0.05), the abortion rate of the PPOS protocol group was higher than that of the ultra-short protocol group. The clinical pregnancy and live birth rates were significantly higher in the PPOS protocol group than in the ultra-short protocol group (p < 0.05). The clinical pregnancy rates in the PPOS protocol group and the ultra-short protocol group were 32.35 % and 25.53 % respectively while the live birth rates were 27.45 % and 21.28 % respectively. ConclusionCompared with the ultra-short protocol, the PPOS protocol improves the number of follicles, the number of eggs, clinical pregnancy, and live birth rates in POR patients. The PPOS protocol could, therefore, provide a novel treatment strategy for inducing ovulation in POR patients.

Effects of estradiol pretreatment during follicular period on outcome of in vitro fertilization and embryo transfer treatment for poor ovarian responder with high FSH level

TO observe the effects of estradiol pretreatment during follicular period on outcomes of in vitro fertilization and embryo transfer (IVF-ET) treatment for poor ovrian responder(POR) with high FSH level. A prospective randomized controlled study. A total of 323 POR with high level who have undergoing IVF-ET treatment were randomly divided into the pretreatment group(n=163) and non-pretreatment group(n=160) acording to whether the estradial pretreatmen (oral administration with 17- β estradiol for 4mg-6mg/d at the second day to the fourth day of menstrual cycle) were conducted before super ovulation induction.General information and indices relevant to the outcome of IVF-ET treatment of two groups were compared. In the pretreatment group, serum follicle-stimulating hormone (FSH)(13.77/14.17IU/ml, p=0.53)levels on the second day of menstruation(D2) and the fifth day(D5) were statistically significant；The D2 serum FSH in the non-pretreatment group was also statistically significant compared to that of the FSH in D5 (13.94/8.85，P=0.00).However,there was no statistical significance in comparing D2 FSH (13.98/13.94IU，P=0.51) and D5 FSH (8.79/8.85IU，P=0.45) values between the two groups.The differences of age (37.93/37.56years,P=0.5),BMI (22.13/21.80Kg/m2,P=0.16),AMH (0.89/0.91ng/ml,P=0.57),basal antral follicle count ( AFC ) (3.57/3.59,P=0.23),the number of retrieved oocytes (2.68/2.25,P=0.70) ,unovulated rate(15.34/13.13,P=0.569), endometrial thickness (10.49/10.49mm，P=0.41),the number of embryos transferred (1.60/1.63,P=0.12),transplant cancellation rate(28.21/31.90%,P=0.474) and clinical pregnancy rate (13.30/11.70%,P=0.10) were not statistically significant between the two groups. Estradiol pretreatment in follicular phase at POR patients with high FSH level did not increase the number of MII eggs rate and clinic pregnancy rate.On the contrary, an increase in the Gn dosage and extended treatment period can impose unnecessary burden on a patient, both financially and mentally. To some extent, the level of FSH only reflects the function of the ovary. Therefore, reducing the blood FSH level cannot increase the number of eggs nor improve the clinical pregnancy outcome.

Potential sperm contributions to the murine zygote predicted by in silico analysis.

Paternal contributions to the zygote are thought to extend beyond delivery of the genome and paternal RNAs have been linked to epigenetic transgenerational inheritance in different species. In addition, sperm-egg fusion activates several downstream processes that contribute to zygote formation, including PLC zeta-mediated egg activation and maternal RNA clearance. Since a third of the preimplantation developmental period in the mouse occurs prior to the first cleavage stage, there is ample time for paternal RNAs or their encoded proteins potentially to interact and participate in early zygotic activities. To investigate this possibility, a bespoke next-generation RNA sequencing pipeline was employed for the first time to characterise and compare transcripts obtained from isolated murine sperm, MII eggs and pre-cleavage stage zygotes. Gene network analysis was then employed to identify potential interactions between paternally and maternally derived factors during the murine egg-to-zygote transition involving RNA clearance, protein clearance and post-transcriptional regulation of gene expression. Our in silico approach looked for factors in sperm, eggs and zygotes that could potentially interact co-operatively and synergistically during zygote formation. At least five sperm RNAs (Hdac11, Fbxo2, Map1lc3a, Pcbp4 and Zfp821) met these requirements for a paternal contribution, which with complementary maternal co-factors suggest a wider potential for extra-genomic paternal involvement in the developing zygote.

HDAC8 functions in spindle assembly during mouse oocyte meiosis

HDAC8 is a class I histone deacetylase that functions in a variety of biological processes through its non-histone substrates. However, its roles during oocyte meiosis remain elusive. Here, we document that HDAC8 localizes at spindle poles and positively participates in the regulation of microtubule organization and spindle assembly in mouse oocytes. Depletion of HDAC8 by siRNA-based gene silencing results in various spindle defects and chromosome misalignment during oocyte meiotic maturation, accompanied by impaired kinetochore-microtubule attachments. Consequently, a higher incidence of aneuploidy is generated in HDAC8-depleted MII eggs. In addition, inhibition of HDAC8 activity with its selective inhibitor PCI-34051 phenocopies the spindle/chromosome defects resulting from HDAC8 depletion by siRNA injection. Finally, we find that HDAC8 is required for the correct localization of ϕ-tubulin to spindle poles. Collectively, these data reveal that HDAC8 plays a significant role in regulating spindle assembly and thus ensuring the euploidy in mouse eggs.

Testicular sperm correlates with a similar rate of euploid embryo development compared to cycles using ejaculated sperm

To evaluate the percentage of euploid embryo development in patients with obstructive and non-obstructive azoospermia. Retrospective data analysis. From March 2013 to April 2016, 36 azoospermic men underwent 45 cycles of ICSI-PGS cycles with blastocyst trophoectoderm biopsy for comprehensive chromosomal screening using high density oligonucleotide microarray comparative genomic hybridization. 30 cycles used testicular sperm from obstructive azoospermia men with prior vasectomy, 9 cycles were non-obstructive, and 6 cycles were from azoospermic men with unknown causes. The results were compared with 621 In-Vitro Fertilization (IVF) patients that used ejaculated sperm during the same time period. 3,475 blastocysts were generated and underwent trophoectoderm biopsy and PGS. Tabled 1Age group# ICSI Cycle# MII eggs ICSI’d#2PN(Fert rate)Blastocyst # (rate)Euploid #(rate)P value<37TESE38446341 (76.5%)206 (60.4%)133/163 (81.6%)P=0.6<37EJ40143723096 (70.8%)3271/5361 (61.0%)1911/2491 (76.7%)38-42TESE75448 (88.5%)19 (39.6%)10/19 (52.5%)P=0.438-42EJ2201044885/1044 (84.8%)771/1537 (50.2%)306/802 (38.2%) Open table in a new tab Our data demonstrate that testicular sperm from azoospermic patients have comparable fertilization, blastocyst formation and euploid rates compared to cycles using ejaculated sperm. These comparable rates of fertilization, blastocyst formation and rates of euploid embryos with the use of testicular sperm requires further study but may be due in part to female partners with more favorable fertility potential.

Characterization of the injection funnel during intracytoplasmic sperm injection reflects cytoplasmic maturity of the oocyte

To quantify cytoplasmic maturity on the basis of intracytoplasmic sperm injection (ICSI) injection funnel manifestation and to evaluate influence factors of the latter. Prospective study. Private fertility center. A total of 31 patients with good ovarian response. Mature and immature oocytes were injected intracytoplasmatically. Formation and persistence of an injection funnel was documented and measured. ICSI funnel size, persistence of injection funnel, rates of degeneration and fertilization, embryo quality. Funnel volume in germinal vesicle stage oocytes (prophase I [PI]) was significantly smaller than that of metaphase I (MI) and MII oocytes. Immature eggs (PI, MI) almost never showed a persistent funnel 2-4minutes after ICSI, whereas in MII eggs the funnel was still observable in 35% (117/334) of the cases. Uni- and multivariate analysis revealed that pipette type and stimulation protocol significantly influenced appearance of injection funnel. Funnel volume in oocytes that fertilized regularly was significantly higher compared with three-polar body and degenerated oocytes. Oocyte maturation within the follicle is closely associated with a remarkable change in cytoplasm viscosity from an aqueous to a more viscous subtype. Precise evaluation of the injection funnel may help to explain deviations from expected ICSI outcome and could also assist in optimizing controlled ovarian hyperstimulation.

Role of Caspase-3 Cleaved IP3R1 on Ca2+Homeostasis and Developmental Competence of Mouse Oocytes and Eggs

Apoptosis in most cell types is accompanied by altered Ca(2+) homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3 R1) generates a 95-kDa C-terminal fragment (C-IP3 R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca(2+) homeostasis and express C-IP3 R1, although whether or not C-IP3 R1 expression contributes to Ca(2+) misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding C-IP3 R1. We found that: (1) expression of C-IP3 R1 in eggs lowered the Ca(2+) content of the endoplasmic reticulum (ER), although, as C-IP3 R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; (2) expression of C-IP3 R1 in eggs enhanced fragmentation associated with aging; (3) endogenous IP3 R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3 R1 in eggs with downregulated IP3 R1 partly restored fragmentation; (4) C-IP3 R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca(2+) pool that occurs during maturation, undermined the Ca(2+) oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3 R1 and C-IP3 R1 in regulating Ca(2+) homeostasis and the ER Ca(2+) content during oocyte maturation. Nevertheless, the role of C-IP3 R1 on Ca(2+) homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3 R1 remains intact and C-IP3 R1 undergoes rapid turnover.

Specificity of calcium/calmodulin-dependent protein kinases in mouse egg activation

CaMKIIγ, the predominant CaMKII isoform in mouse eggs, controls egg activation by regulating cell cycle resumption. In this study we further characterize the involvement and specificity of CaMKIIγ in mouse egg activation. Using exogenous expression of different cRNAs in Camk2g−/− eggs, we show that the other multifunctional CaM kinases, CaMKI, and CaMKIV, are not capable of substituting CaMKIIγ to initiate cell cycle resumption in response to a rise in intracellular Ca2+. Exogenous expression of Camk2g or Camk2d results in activation of nearly 80% of Camk2g−/− MII eggs after stimulation with SrCl2, which does not differ from the incidence of activation of wild-type eggs expressing exogenous Egfp. In contrast, none of the Camk2g−/− MII eggs expressing Camk1 or Camk4 activate in response to SrCl2 treatment. Expression of a constitutively active form of Camk4 (ca-Camk4), but not Camk1, triggers egg activation. EMI2, an APC/C repressor, is a key component in regulating egg activation downstream of CaMKII in both Xenopus laevis and mouse. We show that exogenous expression of either Camk2g, Camk2d, or ca-Camk4, but not Camk1, Camk4, or a catalytically inactive mutant form of CaMKIIγ (kinase-dead) in Camk2g−/− mouse eggs leads to almost complete degradation (~90%) of exogenously expressed EMI2 followed by cell cycle resumption. Thus, degradation of EMI2 following its phosphorylation specifically by CaMKII is mechanistically linked to and promotes cell cycle resumption in MII eggs.

Motherhood after Malignancy: Embracing Oocyte Banking (OB) as a Means to Preserve Fertility between Diagnosis and Cure

and outcomes Age (y) (mean) E2 Day of ov trigger (pg/ml) Total no. retrieved eggs Total no. MII eggs had such a prediction based on age alone. Compared with age-based controls, PredictIVF-D showed 75% improved predictive power, ability to discriminate cases with differential prognoses (10% improvement in area-underthe-curve in a receiver operating characteristic analysis), while extending the dynamic range from four discrete, age-based probabilities to a continuous spectrum ranging from 7 to 60%. Table 1 shows the population distribution and error for five different prognostics groups. CONCLUSION(S): Using a validated, multi-center IVF prediction model, we showed that a large portion of patients with prior IVF would receive a significantly different predicted probability compared with agebased counseling, at improved predictive power and discrimination, and reliable accuracy.

Down-regulation of store-operated Ca2+ entry during mammalian meiosis is required for the egg-to-embryo transition

A specialized Ca(2+) transient at fertilization represents the universal driver for the egg-to-embryo transition. Ca(2+) signaling remodels during oocyte maturation to endow the egg with the capacity to produce the specialized Ca(2+) transient at fertilization, which takes the form of a single (e.g. Xenopus) or multiple (e.g. mouse) Ca(2+) spikes depending on the species. Store-operated Ca(2+) entry (SOCE) is the predominant Ca(2+) influx pathway in vertebrate oocytes, and in Xenopus SOCE completely inactivates during meiosis. Here, we show that SOCE is downregulated during mouse meiosis, but remains active in mature metaphase II eggs. SOCE inhibition is due to a decreased ability of the Ca(2+) sensor STIM1 to translocate to the cortical endoplasmic reticulum domain and due to internalization of Orai1. Reversing SOCE downregulation by overexpression of STIM1 and Orai1 prolongs the Ca(2+) oscillations at egg activation and disrupts the egg-to-embryo transition. Thus, SOCE downregulation during mammalian oocyte maturation is a crucial determinant of the fertilization-specific Ca(2+) transient, egg activation and early embryonic development.

Receptor for activated C kinase (RACK) and protein kinase C (PKC) in egg activation

In somatic cells, translocation of PKCs is facilitated by receptor for activated C kinase (RACK); however its involvement in egg activation is still elusive. We have followed the translocation pattern of conventional and novel PKCs (cPKCs and nPKCs, respectively) upon egg activation. Confocal microscopy indicated the expression and localization of RACK1, a specific receptor protein for cPKCs. Activation of MII eggs, led to translocation to the egg cortex of PKCα, βII and δ and the co-translocation of RACK1, with both PKCα and PKCβII. The association of PKC and actin, both known to be involved in cortical granules exocytosis (CGE) with RACK1, was demonstrated by co-immunoprecipitation. Egg activation resulted in an increased RACK1 level along with a decreased level of PKCβII. Based on these results, we suggest that upon egg activation, RACK1 shuttles activated cPKCs to the egg cortex, thus facilitating CGE.

Localization and Expression of Peroxiredoxin II in the Mouse Ovary, Oviduct, Uterus, and Preimplantation Embryo

Peroxiredoxin (Prx) II belongs to a recently discovered family of peroxidases that play important roles in antioxidation and signal transduction. In this study, we aimed to study the localization and expression of Prx II in the mouse ovary, oviduct, and uterus, and preimplantation embryos. Immunohistochemical staining analysis showed that, in the ovary, Prx II was expressed in the oocyte cytoplasm of the primary follicle, the secondary follicle, and the premature follicle; Prx II was expressed in germinal vesicle-intact oocytes (GV oocytes) and metaphase II eggs (MII eggs), as well as at various stages in early embryos. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that the Prx II mRNA was expressed at a high level in GV eggs, slightly lower levels in MII eggs, and had no detectable expression in four-cell embryos and early blastocysts. In the oviduct, Prx II was expressed in the epithelia, while in the uterus Prx II was mainly distributed in the endometrial stroma. Taken together, our results suggest that Prx II plays a key antioxidation role in the maturation of oocytes and development of early embryos, thus providing crucial experimental evidence for further exploring the function of Prx II in the development of oocytes and preimplantation embryos.

HCO3−/Cl− Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

BackgroundGerminal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO3 −/Cl− exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO3 −/Cl− exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO3 −/Cl− exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established.Methodology/Principal FindingsExpression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO3 −/Cl− exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO3 −/Cl− exchange in mature MII eggs. A GFP-tagged form of the HCO3 −/Cl− exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation.ConclusionsDirect manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO3 −/Cl− exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.